Bioinformatic screening for candidate mutations underlying phenotypic traits in domestic animals

Domestic animals represent excellent model organisms for gene mapping and identification of mutations underlying phenotypic traits. Humans have selected spontaneous mutations in farm and companion animals since they were domesticated and this has resulted in large phenotypic variation among different breeds. In this thesis, we evaluate the candidate mutations in domesticated animals from NGS and SNP genotype data using bioinformatic analysis. Functional significance of coding sequence polymorphisms was assessed using both available bioinformatics resources and in-house pipelines. In consequence, pig and rabbit sequencing revealed major sweeps for genes (NR6A1, LCORL and PLAG1) for body length and increased number of vertebrae in domestic pigs and genes (GRIK2 and SOX2) affecting brain and neuronal development in rabbit domestication. Genome-wide association mapping for demodicosis disease in Staffordshire Bull Terrier dog show several preliminary candidate risk loci (CFA17, 18, 28 and 29) containing interesting candidate genes providing a good basis for further evaluation. Additionally, we also highlight some opportunities and pitfalls of whole genome resequencing using the Ion Proton platform and developed a tool DevRO (using deviant read paired orientation) for detection of large structural variants for NGS data from paired-end sequencing or mate pair. This method will be useful when large numbers of populations are resequenced as compared to traditional methods that can detect the structural variants in a pair wise manner

Bioinformatics analysis of ZBED6, a novel transcription factor in mammals

The identification of a regulatory mutation in the intron 3 of Insulin like growth factor 2, was a major finding. This single nucleotide mutation in the non coding region abrogates the interaction of nuclear factor ZBED6, resulting in the 3-4% increase in the muscle mass of domesticated pigs. The mutation was observed in the evolutionary conserved CpG island that is hypomethylated in skeletal muscle. ZBED6 has been derived from a domesticated DNA transposon exclusively present in the placental mammals. Chromatin immuno precipitation (ChIP) sequencing in mouse C2C12 myoblasts using anti-ZBED6 antibody identified 2499 ZBED6 binding fragments. The de novo search on the binding fragments of ZBED6 showed the consensus sequence of 5´-GCTCG-3´. ZBED6 binding fragments contain more than 1200 genes with annotated functions in biological processes, transcriptional regulation, neurogenesis, cell signaling and muscle development. In present study we have done bioinformatics analysis on the ZBED6 using TRANSFAC database professional version 2010.1 in order to identify the other transcription factors co-regulating the expression of ZBED6 target genes. The ChIP data (ZBED6 target genes) and microarray expression data (siRNA silenced ZBED6) in mouse C2C12 cells were used in this study for finding the binding sites for transcription factors in the promoter regions. The genes associated with ZBED6 showed significant overrepresentation of binding sites of transcription factors SP1, ZF5, E2F1, ZBED6, AP2alpha and KROX in their promoter regions. Majority of factors found have GC rich binding sites and belongs to zinc finger families. The obtained factors show role in tumor suppression. The microarray expression data analysis showed that MEF2 and SRF transcription factors binding sites are significantly present in the promoters of co-pressed genes. The ZBED6 binding sites that were at a distance of 500 kb away from known transcription start site TSS, showed OCT1 and IRF1 binding sites. There is a possibility that these factors are the enhancer elements for many ZBED6 target genes. Few long non-coding RNAs were also identified in the vicinity of ZBED6 binding sites present at a distance of 500 kb away from known TSS

Hop Mice Display Synchronous Hindlimb Locomotion and a Ventrally Fused Lumbar Spinal Cord Caused by a Point Mutation in Ttc26

Identifying the spinal circuits controlling locomotion is critical for unravelling the mechanisms controlling the production of gaits. Development of the circuits governing left-right coordination relies on axon guidance molecules such as ephrins and netrins. To date, no other class of proteins have been shown to play a role during this process. Here, we have analyzed hop mice, which walk with a characteristic hopping gait using their hindlimbs in synchrony. Fictive locomotion experiments suggest that a local defect in the ventral spinal cord contributes to the aberrant locomotor phenotype. Hop mutant spinal cords had severe morphologic defects, including the absence of the ventral midline and a poorly defined border between white and gray matter. The hop mice represent the first model where, exclusively found in the lumbar domain, the left and right components of the central pattern generators (CPGs) are fused with a synchronous hindlimb gait as a functional consequence. These defects were associated with abnormal developmental processes, including a misplaced notochord and reduced induction of ventral progenitor domains. Whereas the underlying mutation in hop mice has been suggested to lie within the Ttc26 gene, other genes in close vicinity have been associated with gait defects. Mouse embryos carrying a CRISPR replicated point mutation within Ttc26 displayed an identical morphologic phenotype. Thus, our data suggest that the assembly of the lumbar CPG network is dependent on fully functional TTC26 protein

A frame-shift mutation in COMTD1 is associated with impaired pheomelanin pigmentation in chicken

Author summaryVertebrates possess two types of melanin, red/yellow pheomelanin and black/brown eumelanin. In this study, we report that the recessive Inhibitor of gold phenotype in chicken, which causes a severe defect in pheomelanin pigmentation, is associated with a mutation that most likely inactivates the COMTD1 gene. This gene encodes an O-methyltransferase enzyme and is present throughout vertebrate evolution, but is one of the many genes in vertebrate genomes for which the biological function is still poorly understood. This is the first report of a COMTD1 mutation associated with a phenotypic effect. We show that the COMTD1 protein is present in mitochondria in pigment cells. Furthermore, inactivation of the gene in a mouse pigment cell line results in a significant reduction in metabolites that are important for the synthesis of pheomelanin. We hypothesize that COMTD1 activity protects pigment cells from oxidative stress and that inactivation of this function impairs the production of pheomelanin. It is likely that COMTD1 has a similar function in other cell types. This study establishes this chicken mutation as a model for further studies of COMTD1 function.The biochemical pathway regulating the synthesis of yellow/red pheomelanin is less well characterized than the synthesis of black/brown eumelanin. Inhibitor of gold (IG phenotype) is a plumage colour variant in chicken that provides an opportunity to further explore this pathway since the recessive allele (IG) at this locus is associated with a defect in the production of pheomelanin. IG/IG homozygotes display a marked dilution of red pheomelanin pigmentation, whilst black pigmentation (eumelanin) is only slightly affected. Here we show that a 2-base pair insertion (frame-shift mutation) in the 5(th) exon of the Catechol-O-methyltransferase containing domain 1 gene (COMTD1), expected to cause a complete or partial loss-of-function of the COMTD1 enzyme, shows complete concordance with the IG phenotype within and across breeds. We show that the COMTD1 protein is localized to mitochondria in pigment cells. Knockout of Comtd1 in a mouse melanocytic cell line results in a reduction in pheomelanin metabolites and significant alterations in metabolites of glutamate/glutathione, riboflavin, and the tricarboxylic acid cycle. Furthermore, COMTD1 overexpression enhanced cellular proliferation following chemical-induced transfection, a potential inducer of oxidative stress. These observations suggest that COMTD1 plays a protective role for melanocytes against oxidative stress and that this supports their ability to produce pheomelanin

Feasibility to use whole-genome sequencing as a sole diagnostic method to detect genomic aberrations in pediatric B-cell acute lymphoblastic leukemia

IntroductionThe suitability of whole-genome sequencing (WGS) as the sole method to detect clinically relevant genomic aberrations in B-cell acute lymphoblastic leukemia (ALL) was investigated with the aim of replacing current diagnostic methods.MethodsFor this purpose, we assessed the analytical performance of 150 bp paired-end WGS (90x leukemia/30x germline). A set of 88 retrospective B-cell ALL samples were selected to represent established ALL subgroups as well as ALL lacking stratifying markers by standard-of-care (SoC), so-called B-other ALL.ResultsBoth the analysis of paired leukemia/germline (L/N)(n=64) as well as leukemia-only (L-only)(n=88) detected all types of aberrations mandatory in the current ALLTogether trial protocol, i.e., aneuploidies, structural variants, and focal copy-number aberrations. Moreover, comparison to SoC revealed 100% concordance and that all patients had been assigned to the correct genetic subgroup using both approaches. Notably, WGS could allocate 35 out of 39 B-other ALL samples to one of the emerging genetic subgroups considered in the most recent classifications of ALL. We further investigated the impact of high (90x; n=58) vs low (30x; n=30) coverage on the diagnostic yield and observed an equally perfect concordance with SoC; low coverage detected all relevant lesions.DiscussionThe filtration of the WGS findings with a short list of genes recurrently rearranged in ALL was instrumental to extract the clinically relevant information efficiently. Nonetheless, the detection of DUX4 rearrangements required an additional customized analysis, due to multiple copies of this gene embedded in the highly repetitive D4Z4 region. We conclude that the diagnostic performance of WGS as the standalone method was remarkable and allowed detection of all clinically relevant genomic events in the diagnostic setting of B-cell ALL

Defining post-acute COVID-19 syndrome (PACS) by an epigenetic biosignature in peripheral blood mononuclear cells

Post-acute COVID-19 syndrome (PACS) has been defined as symptoms persisting after clearance of a COVID-19 infection. We have previously demonstrated that alterations in DNA methylation (DNAm) status persist in individuals who recovered from a COVID-19 infection, but it is currently unknown if PACS is associated with epigenetic changes. We compared DNAm patterns in patients with PACS with those in controls and in healthy COVID-19 convalescents and found a unique DNAm signature in PACS patients. This signature unravelled modified pathways that regulate angiotensin II and muscarinic receptor signalling and protein–protein interaction networks that have bearings on vesicle formation and mitochondrial function

Whole-Genome Sequencing of a Canine Family Trio Reveals a FAM83G Variant Associated with Hereditary Footpad Hyperkeratosis

Over 250 Mendelian traits and disorders, caused by rare alleles have been mapped in the canine genome. Although each disease is rare in the dog as a species, they are collectively common and have major impact on canine health. With SNP-based genotyping arrays, genome-wide association studies (GWAS) have proven to be a powerful method to map the genomic region of interest when 10-20 cases and 10-20 controls are available. However, to identify the genetic variant in associated regions, fine-mapping and targeted re-sequencing is required. Here we present a new approach using whole-genome sequencing (WGS) of a family trio without prior GWAS. As a proof-of-concept, we chose an autosomal recessive disease known as hereditary footpad hyperkeratosis (HFH) in Kromfohrl änder dogs. To our knowledge, this is the first time this family trio WGS-approach, has successfully been used to identify a genetic variant that perfectly segregates with a canine disorder. The sequencing of three Kromfohrl änder dogs from a family trio (an affected offspring and both its healthy parents) resulted in an average genome coverage of 9.2X per individual. After applying stringent filtering criteria for candidate causative coding variants, 527 single nucleotide variants (SNVs) and 15 indels were found to be homozygous in the affected offspring and heterozygous in the parents. Using the computer software packages ANNOVAR and SIFT to functionally annotate coding sequence differences and to predict their functional effect, resulted in seven candidate variants located in six different genes. Of these, only FAM83G:c155G>C (p.R52P) was found to be concordant in eight additional cases and 16 healthy Kromfohrl änder dogs

The spectrum of tuberculosis described as differential DNA methylation patterns in alveolar macrophages and alveolar T cells

Background: Host innate immune cells have been identified as key players in the early eradication of Mycobacterium tuberculosis and in the maintenance of an anti-mycobacterial immune memory, which we and others have shown are induced through epigenetic reprogramming. Studies on human tuberculosis immunity are dominated by those using peripheral blood as surrogate markers for immunity. We aimed to investigate DNA methylation patterns in immune cells of the lung compartment by obtaining induced sputum from M. tuberculosis- exposed subjects including symptom-free subjects testing positively and negatively for latent tuberculosis as well as patients diagnosed with active tuberculosis. Alveolar macrophages and alveolar T cells were isolated from the collected sputum and DNA methylome analyses performed (Illumina Infinium Human Methylation 450 k).Results: Multidimensional scaling analysis revealed that DNA methylomes of cells from the tuberculosis-exposed subjects and controls appeared as separate clusters. The numerous genes that were differentially methylated between the groups were functionally connected and overlapped with previous findings of trained immunity and tuberculosis. In addition, analysis of the interferon-gamma release assay (IGRA) status of the subjects demonstrated that the IGRA status was reflected in the DNA methylome by a unique signature.Conclusions: This pilot study suggests that M. tuberculosis induces epigenetic reprogramming in immune cells of the lung compartment, reflected as a specific DNA methylation pattern. The DNA methylation signature emerging from the comparison of IGRA-negative and IGRA-positive subjects revealed a spectrum of signature strength with the TB patients grouping together at one end of the spectrum, both in alveolar macrophages and T cells. DNA methylation-based biosignatures could be considered for further development towards a clinically useful tool for determining tuberculosis infection status and the level of tuberculosis exposure.Funding Agencies|Linkoeping University; Forskningsradet Sydoestra Sverige; Swedish Research Council [FORSS-932096]; Swedish Heart Lung Foundation [2015-02593, 2018-02961, 2018-04246, 106-2018-FONDECYT]; CONCYTEC-PROCIENCIA [2018-05973, 20150709]; Board of Research at the Karolinska Institute, Stockholm [20180613]; World Infection Fund</p

Epigenetic rewiring of pathways related to odour perception in immune cells exposed to SARS-CoV-2 in vivo and in vitro

A majority of SARS-CoV-2 recoverees develop only mild-to-moderate symptoms, while some remain completely asymptomatic. Although viruses, including SARS-CoV-2, may evade host immune responses by epigenetic mechanisms including DNA methylation, little is known about whether these modifications are important in defence against and healthy recovery from COVID-19 in the host. To this end, epigenome-wide DNA methylation patterns from COVID-19 convalescents were compared to uninfected controls from before and after the pandemic. Peripheral blood mononuclear cell (PBMC) DNA was extracted from uninfected controls, COVID-19 convalescents, and symptom-free individuals with SARS-CoV-2-specific T cell-responses, as well as from PBMCs stimulated in vitro with SARS-CoV-2. Subsequently, the Illumina MethylationEPIC 850K array was performed, and statistical/bioinformatic analyses comprised differential DNA methylation, pathway over-representation, and module identification analyses. Differential DNA methylation patterns distinguished COVID-19 convalescents from uninfected controls, with similar results in an experimental SARS-CoV-2 infection model. A SARS-CoV-2-induced module was identified in vivo, comprising 66 genes of which six (TP53, INS, HSPA4, SP1, ESR1, and FAS) were present in corresponding in vitro analyses. Over-representation analyses revealed involvement in Wnt, muscarinic acetylcholine receptor signalling, and gonadotropin-releasing hormone receptor pathways. Furthermore, numerous differentially methylated and network genes from both settings interacted with the SARS-CoV-2 interactome. Altered DNA methylation patterns of COVID-19 convalescents suggest recovery from mild-to-moderate SARS-CoV-2 infection leaves longstanding epigenetic traces. Both in vitro and in vivo exposure caused epigenetic modulation of pathways thataffect odour perception. Future studies should determine whether this reflects host-induced protective antiviral defense or targeted viral hijacking to evade host defence.Funding Agencies: Swedish Heart and Lung Foundation [20200319, 20200067, 20210067]; Swedish Research Council [Covid-19/biobank 210202]; Open Medicine foundation [OMF190626]; Hjärt-Lungfonden; Open Medicine Foundation; Vetenskapsrådet</p

Additional file 2: of Evaluation of whole-genome sequencing of four Chinese crested dogs for variant detection using the ion proton system

PI chip productivity and per base quality scores. Table describing the yield and base quality of eight sequencing chips. (XLSX 40ย�kb